A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

AIMS: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). METHODS: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. RESULTS: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. CONCLUSIONS: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.

New diketopiperazines as vectors for peptide protection and brain delivery: Synthesis and biological evaluation.

New strategies allowing the transfer of molecules, especially peptides, through the blood-brain barriers are a major pharmacological challenge for the treatment of brain diseases. The present study aims at evaluating in vivo the cerebral bioavailability of carrier systems, based on small and functionalizable 2,5-diketopiperazine (DKP) motifs. We studied 2 different cyclo(Lys-Lys) DKP scaffolds alone and a cyclo(Lys-Gly) DKP carrier bearing as peptide model, the tau protein hexapeptide VQIVYK sequence. The different carrier systems were synthesized and radiolabeled using one of the free domains. The stability, biodistribution, and ability to cross blood-brain barrier were investigated in vivo in mice for 99m Tc-DKP scaffolds, 99m Tc-HVQIVYK peptide alone, and 99m Tc-DKP-VQIVYK. 125 I-labelled bovine serum albumin was used as negative control for brain uptake. Both radiolabeled DKPs scaffolds and 99m Tc-DKP-VQIVYK showed a high stability, while peptide 99m Tc-HVQIVYK alone was quickly degraded in vivo. The presence of 99m Tc-DKPs scaffolds and 99m Tc-DKP-VQIVYK was observed in the ventricular and subarachnoid spaces and to a lower extent in the brain parenchyma up to 45 minutes post-injection in mice. This work highlights the potentiality of DKP scaffolds as vectors to transport peptides into the brain by limiting proteolysis and favoring cerebral bioavailability.

Melatonin reduces excitotoxic blood-brain barrier breakdown in neonatal rats.

The blood-brain barrier (BBB) is a complex structure that protects the central nervous system from peripheral insults. Understanding the molecular basis of BBB function and dysfunction holds significant potential for future strategies to prevent and treat neurological damage. The aim of our study was (1) to investigate BBB alterations following excitotoxicity and (2) to test the protective properties of melatonin. Ibotenate, a glutamate analog, was injected intracerebrally in postnatal day 5 (P5) rat pups to mimic excitotoxic injury. Animals were than randomly divided into two groups, one receiving intraperitoneal (i.p.) melatonin injections (5mg/kg), and the other phosphate buffer saline (PBS) injections. Pups were sacrificed 2, 4 and 18 h after ibotenate injection. We determined lesion size at 5 days by histology, the location and organization of tight junction (TJ) proteins by immunohistochemical studies, and BBB leakage by dextran extravasation. Expression levels of BBB genes (TJs, efflux transporters and detoxification enzymes) were determined in the cortex and choroid plexus by quantitative PCR. Dextran extravasation was seen 2h after the insult, suggesting a rapid BBB breakdown that was resolved by 4h. Extravasation was significantly reduced in melatonin-treated pups. Gene expression and immunohistochemical assays showed dynamic BBB modifications during the first 4h, partially prevented by melatonin. Lesion-size measurements confirmed white matter neuroprotection by melatonin. Our study is the first to evaluate BBB structure and function at a very early time point following excitotoxicity in neonates. Melatonin neuroprotects by preventing TJ modifications and BBB disruption at this early phase, before its previously demonstrated anti-inflammatory, antioxidant and axonal regrowth-promoting effects.

Physiology of blood-brain interfaces in relation to brain disposition of small compounds and macromolecules.

The brain develops and functions within a strictly controlled environment resulting from the coordinated action of different cellular interfaces located between the blood and the extracellular fluids of the brain, which include the interstitial fluid and the cerebrospinal fluid (CSF). As a correlate, the delivery of pharmacologically active molecules and especially macromolecules to the brain is challenged by the barrier properties of these interfaces. Blood-brain interfaces comprise both the blood-brain barrier located at the endothelium of the brain microvessels and the blood-CSF barrier located at the epithelium of the choroid plexuses. Although both barriers develop extensive surface areas of exchange between the blood and the neuropil or the CSF, the molecular fluxes across these interfaces are tightly regulated. Cerebral microvessels acquire a barrier phenotype early during cerebral vasculogenesis under the influence of the Wnt/ß-catenin pathway, and of recruited pericytes. Later in development, astrocytes also play a role in blood-brain barrier maintenance. The tight choroid plexus epithelium develops very early during embryogenesis. It is specified by various signaling molecules from the embryonic dorsal midline, such as bone morphogenic proteins, and grows under the influence of Sonic hedgehog protein. Tight junctions at each barrier comprise a distinctive set of claudins from the pore-forming and tightening categories that determine their respective paracellular barrier characteristics. Vesicular traffic is limited in the cerebral endothelium and abundant in the choroidal epithelium, yet without evidence of active fluid phase transcytosis. Inorganic ion transport is highly regulated across the barriers. Small organic compounds such as nutrients, micronutrients and hormones are transported into the brain by specific solute carriers. Other bioactive metabolites, lipophilic toxic xenobiotics or pharmacological agents are restrained from accumulating in the brain by several ATP-binding cassette efflux transporters, multispecific solute carriers, and detoxifying enzymes. These various molecular effectors differently distribute between the two barriers. Receptor-mediated endocytotic and transcytotic mechanisms are active in the barriers. They enable brain penetration of selected polypeptides and proteins, or inversely macromolecule efflux as it is the case for immnoglobulins G. An additional mechanism specific to the BCSFB mediates the transport of selected plasma proteins from blood into CSF in the developing brain. All these mechanisms could be explored and manipulated to improve macromolecule delivery to the brain.

[Biological roles of trace elements in the brain with special focus on Zn and Fe]. / Rôles biologiques des éléments traces dans le cerveau--exemples du Zn et du Fe.

INTRODUCTION: Many metals like iron (Fe), copper (Cu) or zinc (Zn) fulfil various essential biological functions and are thus vital for all living organisms. For instance, they play important roles in nervous tissue, participating in a wide range of processes such as neurotransmitter synthesis, myelination or synaptic transmission. STATE OF THE ART: As in other tissues, brain cells tightly control the concentration of metals but any excess or deficit can lead to deleterious responses and alter cognitive functions. Of note, certain metals such as Zn, Fe or Cu accumulate in specific brain structures over lifespan and several neurodegenerative diseases are associated with a dysregulation of the homeostatic mechanisms controlling the concentration of these cations. CONCLUSION AND PERSPECTIVES: This review will address some of the cellular and molecular processes controlling the entry and distribution of selected metals (mainly Zn and Fe) in the brain, as well as their roles in synaptic transmission, in the pathogenesis of some neurologic diseases such as Parkinson's disease and Alzheimer's disease, and their impact on cognitive functions.

Blood-brain interfaces and cerebral drug bioavailability.

The low cerebral bioavailability of various drugs is a limiting factor in the treatment of neurological diseases. The restricted penetration of active compounds into the brain is the result of the same mechanisms that are central to the maintenance of brain extracellular fluid homeostasis, in particular from the strict control imposed on exchanges across the blood-brain interfaces. Direct drug entry into the brain parenchyma occurs across the cerebral microvessel endothelium that forms the blood-brain barrier. In addition, local drug concentration measurements and cerebral imaging have clearly shown that the choroid plexuses - the main site of the blood-cerebrospinal fluid (CSF) barrier - together with the CSF circulatory system also play a significant role in setting the cerebral bioavailability of drugs and contrast agents. The entry of water-soluble therapeutic compounds into the brain is impeded by the presence of tight junctions that seal the cerebral endothelium and the choroidal epithelium. The cerebral penetration of many of the more lipid-soluble molecules is also restricted by various classes of efflux transporters that are differently distributed among both blood-brain interfaces, and comprise either multidrug resistance proteins of the ATP-binding cassette superfamily or transporters belonging to several solute carrier families. Expression of these transporters is regulated in various pathophysiological situations, such as epilepsy and inflammation, with pharmacological consequences that have yet to be clearly elucidated. As for brain tumour treatments, their efficacy may be affected not only by the intrinsic resistance of tumour cells, but also by endothelial efflux transporters which exert an even greater impact than the integrity of the endothelial tight junctions. Relevant to paediatric neurological treatments, both blood-brain interfaces are known to develop a tight phenotype very early on in postnatal development, but the developmental profile of efflux transporters still needs to be assessed in greater detail. Finally, the exact role of the ependyma and pia-glia limitans in controlling drug exchanges between brain parenchyma and CSF deserves further attention to allow more precise predictions of cerebral drug disposition and therapeutic efficacy.

Blood-brain interfaces and bilirubin-induced neurological diseases.

The endothelium of the brain microvessels and the choroid plexus epithelium form highly specialized cellular barriers referred to as blood-brain interfaces through which molecular exchanges take place between the blood and the neuropil or the cerebrospinal fluid, respectively. Within the brain, the ependyma and the pia-glia limitans modulate exchanges between the neuropil and the cerebrospinal fluid. All these interfaces are key elements of neuroprotection and fulfill trophic functions; both properties are critical to harmonious brain development and maturation. By analogy to hepatic bilirubin detoxification pathways, we review the transport and metabolic mechanisms which in all these interfaces may participate in the regulation of bilirubin cerebral bioavailability in physiologic conditions, both in adult and in developing brain. We specifically address the role of ABC and OATP transporters, glutathione-S-transferases, and the potential involvement of glucuronoconjugation and oxidative metabolic pathways. Regulatory mechanisms are explored which are involved in the induction of these pathways and represent potential pharmacological targets to prevent bilirubin accumulation into the brain. We then review the possible alteration of the neuroprotective and trophic barrier functions in the course of bilirubin-induced neurological dysfunctions resulting from hyperbilirubinemia. Finally, we highlight the role of the blood-brain and blood-CSF barriers in regulating the brain biodisposition of candidate drugs for the treatment or prevention of bilirubin-induced brain injury.

Regional acetylcholinesterase activity and its correlation with behavioral performances in 15-month old transgenic mice expressing the human C99 fragment of APP.

In addition to Abeta plaques and neurofibrillary tangles, Alzheimer's disease (AD) is characterized by increased brain levels of APP C-terminal fragments. In the present investigation, the cholinergic innervation in forebrain regions of transgenic mice (Tg13592) expressing the human betaAPP C99 fragment was compared to that of non-transgenic controls by measuring the activity of the non-specific catabolic enzyme, acetylcholinesterase (AChE). The AchE activity of Tg13592 mice was altered in several regions implicated in the functional loop of regulation between septum and hippocampus, vulnerable in Alzheimer pathology and critically involved in cognitive functions. In particular, AChE activity was upregulated in three basal forebrain regions containing cholinergic cell bodies, prelimbic cortex, anterior subiculum, and paraventricular thalamus, but downregulated in lateral septum and reticular thalamus. The increased activity in medial septum and anterior subiculum was linearly correlated with poor performances in a spatial learning task, possibly due to cell stress mechanisms. Because of some similarities in terms of neurochemistry and behavior, this mouse model may be of use for studying prodromal AD.

[The choroid plexuses: a dynamic interface between the blood and the cerebrospinal fluid]. / Les plexus choroïdes: une interface dynamique entre sang et liquide cephalo-rachidien.

The choroid plexuses form one of the interfaces that control the brain microenvironment by regulating the exchanges between the blood and the central nervous system. They appear early during brain development. Originating from four different areas of the neural tube, they protrude into the ventricular system of the brain. The choroidal mechanisms involved in the control of brain homeostasis include the structural properties of the epithelial cells that restrict diffusional processes, as well as specific exchange and secretion mechanisms. In addition to the anatomical and histological organization of the choroidal tissue, this review describes the mechanism of cerebrospinal fluid secretion which is the most studied function of the choroid plexus. Experimental evidence for an implication of the choroid plexuses in neuroprotective mechanisms and in the supply of biologically active polypeptides to the brain are also reviewed.

Brain drug delivery, drug metabolism, and multidrug resistance at the choroid plexus.

The choroid plexuses (CPs) have the capability to modulate drug delivery to the cerebrospinal fluid (CSF) and to participate in the overall cerebral biodisposition of drugs. The specific morphological properties of the choroidal epithelium and the existence of a CSF pathway for drug distribution to different targets in the central nervous system suggest that the CP-CSF route is more significant than previously thought for brain drug delivery. In contrast to its role in CSF penetration of drugs, CP is also involved in brain protection in that it has the capacity to clear the CSF from numerous potentially harmful CSF-borne exogenous and endogenous organic compounds into the blood. Furthermore, CP harbors a large panel of drug-metabolizing enzymes as well as transport proteins of the multidrug resistance phenotype, which modulate the cerebral bioavailability of drugs and toxins. The use of an in vitro model of the choroidal epithelium suitable for drug transport studies has allowed the demonstration of the choroidal epithelium acting as an effective metabolic blood-CSF barrier toward some xenobiotics, and that a vectorial, blood-facing efflux of conjugated metabolites occurs at the choroidal epithelium. This efflux involves a specific transporter with characteristics similar to those of the multidrug resistance associated protein (MRP) family members. Indeed, at least one member, MRP1, is largely expressed at the CP epithelium, and localizes at the basolateral membrane. These metabolic and transport features of the choroidal epithelium point out the CP as a major detoxification site within the brain.

Choroid plexus in the central nervous system: biology and physiopathology.

Choroid plexuses (CPs) are localized in the ventricular system of the brain and form one of the interfaces between the blood and the central nervous system (CNS). They are composed of a tight epithelium responsible for cerebrospinal fluid secretion, which encloses a loose connective core containing permeable capillaries and cells of the lymphoid lineage. In accordance with its peculiar localization between 2 circulating fluid compartments, the CP epithelium is involved in numerous exchange processes that either supply the brain with nutrients and hormones, or clear deleterious compounds and metabolites from the brain. Choroid plexuses also participate in neurohumoral brain modulation and neuroimmune interactions, thereby contributing greatly in maintaining brain homeostasis. Besides these physiological functions, the implication of choroid plexuses in pathological processes is increasingly documented. In this review, we focus on some of the novel aspects of CP functions in relation to brain development, transfer of neuro-humoral information, brain/immune system interactions, brain aging, and cerebral pharmaco-toxicology.

In vitro evidence that beta-amyloid peptide 1-40 diffuses across the blood-brain barrier and affects its permeability.

Beta amyloid peptides are major insoluble constituents of amyloid fibrils in senile plaques and cerebrovascular deposits, both characteristic of Alzheimer disease (AD). Low concentrations of soluble forms of amyloid peptides are also present in normal CSF. We previously demonstrated that the 40 amino acid form of soluble beta-amyloid peptide (sAbeta) is rapidly cleared from rat CSF into blood. Herein we hypothesized that a saturable, outwardly directed flux of this peptide occurs at the blood-brain barrier (BBB) and tested whether supraphysiological (possibly pathological) concentrations of sAbeta could alter the permeability of this barrier to a paracellular tracer, polyethylene glycol (PEG). Using an in vitro model of BBB, we showed that influx and efflux of sAbeta were equal, modest (60%-160% greater than that of PEG), and not saturable. These observations suggest that sAbeta moved across the monolayer by a diffusional process, and not via a transporter. PEG flux was doubled immediately after the luminal concentration of cold sAbeta was raised to 5 microM, and was doubled 150 min after the abluminal concentration of sAbeta was increased to 5 microM. Pathological elevations of sAbeta concentration in plasma or brain interstitial fluid may, therefore, alter the permeability of brain capillaries in vivo.

Symposium overview: the role of glutathione in neuroprotection and neurotoxicity.

Although the cytoprotective effects of glutathione (GSH) are well established, additional roles for GSH in brain function are being identified that provide a pharmacological basis for the relationship between alterations in GSH homeostasis and the development of certain neurodegenerative processes. Thus, GSH and glutathione disulfide (GSSG) appear to play important functional roles in the central nervous system (CNS). A symposium, focussing on the emerging science of the roles of GSH in the brain, was held at the 37th annual meeting of the Society of Toxicology, with the emphasis on the role of glutathione in neuroprotection and neurotoxicity. Jean Francois Ghersi-Egea opened the symposium by describing the advances in our understanding of the role of the blood-brain and blood-cerebral spinal fluid (CSF) barriers in either limiting or facilitating the access of xenobiotics into the brain. Once within the brain, a multitude of factors will determine whether a chemical causes toxicity and at which sites such toxicity will occur. In this respect, it is becoming increasingly clear that GSH and its various conjugation enzymes are not evenly distributed throughout the brain. Martin Philbert discussed how this regional heterogeneity might provide a potential basis for the theory of differential sensitivity to neurotoxicants, in various regions of the brain. For certain chemicals, GSH provides neuroprotection, and Edward Lock discussed the selective toxicity of 2-chloropropionic acid (CPA) to the cerebellum and how its modification by modulating brain thiol status provides an example of GSH acting in neuroprotection. The sensitivity of the cerebellum to CPA may also be linked to the ability of this compound to activate a sub-type of the NMDA receptor. Thus, GSH and cysteine alone, or perhaps as conjugates with xenobiotics, may play a role in excitotoxicity via NMDA receptor activation. In contrast, certain chemicals may be converted to neurotoxicants following conjugation with GSH, and Arthur Cooper described how the pyridoxal 5'-phosphate-dependent, cysteine conjugate beta-lyases might predispose the brain to chemical injury in a GSH-dependent manner. The theme of GSH as a potential mediator of chemical-induced neurotoxicity was extended by Terrence Monks, who presented evidence for a role for GSH conjugation in (+/-)-3,4- methylenedioxyamphetamine-mediated serotonergic neurotoxicity.

Demonstration of a coupled metabolism-efflux process at the choroid plexus as a mechanism of brain protection toward xenobiotics.

Brain homeostasis depends on the composition of both brain interstitial fluid and CSF. Whereas the former is largely controlled by the blood-brain barrier, the latter is regulated by a highly specialized blood-CSF interface, the choroid plexus epithelium, which acts either by controlling the influx of blood-borne compounds, or by clearing deleterious molecules and metabolites from CSF. To investigate mechanisms of brain protection at the choroid plexus, the blood-CSF barrier was reconstituted in vitro by culturing epithelial cells isolated from newborn rat choroid plexuses of either the fourth or the lateral ventricle. The cells grown in primary culture on semipermeable membranes established a pure polarized monolayer displaying structural and functional barrier features, (tight junctions, high electric resistance, low permeability to paracellular markers) and maintaining tissue-specific markers (transthyretin) and specific transporters for micronutriments (amino acids, nucleosides). In particular, the high enzymatic drug metabolism capacity of choroid plexus was preserved in the in vitro blood-CSF interface. Using this model, we demonstrated that choroid plexuses can act as an absolute blood-CSF barrier toward 1-naphthol, a cytotoxic, lipophilic model compound, by a coupled metabolism-efflux mechanism. This compound was metabolized in situ via uridine diphosphate glururonosyltransferase-catalyzed conjugation, and the cellular efflux of the glucurono-conjugate was mediated by a transporter predominantly located at the basolateral, i.e., blood-facing membrane. The transport process was temperature-dependent, probenecid-sensitive, and recognized other glucuronides. Efflux of 1-naphthol metabolite was inhibited by intracellular glutathione S-conjugates. This metabolism-polarized efflux process adds a new facet to the understanding of the protective functions of choroid plexuses.

Regional brain variations of cytochrome oxidase activity and motor coordination in Lurcher mutant mice.

Lurcher mutant mice are characterized by massive degeneration of cerebellar Purkinje cells and granule cells and by deficits in motor coordination. Regional brain variations of cytochrome oxidase (CO) activity were analyzed to identify those brain regions with abnormal metabolic activity as a secondary consequence of the cerebellar atrophy and to establish the relationship between CO activity and motor deficits. Lurcher mutants had higher CO activity in all three cerebellar deep nuclei than normal littermate controls of the same background strain. Higher CO activity was also found in Lurcher mutants in brain regions directly connected to the cerebellum, such as the lateral vestibular nucleus, the cochlear nucleus, the red nucleus, the ventrolateral thalamus, the dorsal raphe, the interpeduncular nucleus, and the inferior colliculus. By contrast, there was a sharp decrease in CO activity in the inferior olive. As for brain regions not directly connected to the cerebellum, higher CO activity was observed in the trigeminal motor nucleus and the CA1 molecular layer of the hippocampus, which highlights probable transsynaptic alterations as a secondary consequence of cerebellar atrophy. A positive correlation between CO activity in the red nucleus and latencies before falling in two motor-coordination tests indicates that a compensatory increase of metabolic activity in a cerebellar efferent region is associated with improved behavior.

Electronic spin resonance detection of superoxide and hydroxyl radicals during the reductive metabolism of drugs by rat brain preparations and isolated cerebral microvessels.

A spin trapping technique was used to analyze by electron spin resonance (ESR) the formation of oxygen-derived free radicals during the cerebral reductive metabolism of xenobiotics able to undergo a single electron reduction, i.e. quinones, pyridinium compounds and nitroheterocyclics. Paraquat, menadione and nitrofurazone were used as model compounds of these three classes of molecules. ESR spectra indicative of superoxide and hydroxyl radical formation were obtained by incubation of brain homogenates directly within the ESR cavity at 37 degrees C for each of the three molecules tested. These signals were dependent on nucleotide cofactors, and increased in a time-dependent manner. The NADPH and NADH dependent free radical production was further characterized in brain microsomal and mitochondrial fractions, respectively. By using various combinations of reactive species inactivating enzymes (superoxide dismutase, catalase), a metal chelator (deferoxamine), and an hydroxyl trapping agent (dimethylsulfoxide), it was shown that (1) the primary radical generated was the superoxide anion; and (2) a significant production of the hydroxyl radical also occurred, that was secondary to the superoxide anion production. Consistent signals indicative of the production of both oxygen-derived free radicals were obtained when isolated cerebral microvessels which constitute the blood-brain barrier were incubated with the model molecules. This is of particular toxicological relevance, because this barrier represents a key element in the protection of the brain, and is in close contact with blood-born exogenous molecules.

Drug metabolism in in vitro organotypic and cellular models of mammalian central nervous system: activities of membrane-bound epoxide hydrolase and NADPH-cytochrome P-450 (c) reductase.

The membrane-bound form of epoxide hydrolase and NADPH-cytochrome P-450 (c) reductase are two important enzymes involved in the bioactivation/bioinactivation balance of cerebral tissue. In vivo, the developmental profiles and regional localizations of these two enzymes were investigated in the rat. The regional distribution study showed that they are ubiquitously present among the major brain structures. Both enzyme activities were present in the brain prior to birth, and hence tissue from early developmental stages is suitable to develop in vitro cellular or organotypic models for toxicity studies involving these metabolic pathways. Because various neurotoxicological effects can be dependent on spatio-temporally regulated cell-cell interactions, we aimed to employ organotypic tissue cultures in which the cytoarchitecture was well preserved. In such cultures, the temporal expression profiles of epoxide hydrolase and NADPH cytochrome(c) P-450 reductase reflected the in vivo situation. The technically less demanding pure neuronal and glial cell cultures were also investigated. Detoxification of benzopyrene-4,5-epoxide and superoxide production arising from the reductive metabolism of various drugs were determined in all three systems. The results indicate that though organotypic culture is a good model for the metabolic pathways studied, less complicated single cell cultures can also represent appropriate model systems, providing that the expression of the enzymes involved has been first established in these systems. NADPH-cytochrome P-450 reductase-dependent metabolism is active in both neuronal and glial cells, whereas the detoxification of reactive epoxides occurs mainly in glia.

Capacity of rat brain to metabolize m-dinitrobenzene: an in vitro study.

m-dinitrobenzene (m-DNB) is a neurotoxin producing selective brain lesions, but the in situ metabolic fate of m-DNB in brain is unknown. In this study, nitroreductive capacity of brain towards m-dinitrobenzene (m-DNB) has been investigated. Tissue slices from F344 rat brain stem, forebrain, and liver were separately incubated with 0.2 mM m-DNB. m-DNB and its metabolites were detected by HPLC, and identified by either HPLC or Mass Spectrometry (MS). All three types of tissues showed metabolic activity towards m-DNB. Metabolic disposal of m-DNB was 1.05 +/- 0.11 mumol/g wet weight/h in liver, 0.49 +/- 0.05 in brain stem, and 0.44 +/- 0.05 in forebrain (mean +/- SD, n = 4). m-Nitroaniline was found to be the main metabolite produced by both brain and liver slices, representing 57-66% of the disposal of m-DNB. Liver slices also produced 2(or 4)-amino-4(or 2)-nitrophenol, which was not detected in brain slices. We detected nitrosonitrobenzene in the slices from both parts of brain, but not in liver slices. The glucose consumption of brain slices from both areas were significantly increased in the presence of m-DNB: by 26% in the brain stem (p < 0.001) and by 17.9% in cerebral cortex (p < 0.01). This may be considered a pre-cytotoxic effect. The results demonstrate that brain has considerable nitroreductive capacity towards m-DNB, and that in situ reduction of m-DNB may be responsible for its neurotoxicity.

The rapid flow of cerebrospinal fluid from ventricles to cisterns via subarachnoid velae in the normal rat.

14C-sucrose in 0.5 microliter of buffered saline was infused over 30 sec into one lateral ventricle, and its subsequent distribution was determined in brain, meninges, cerebral blood vessels, and cerebrospinal fluid (CSF) by quantitative autoradiography. Within 3.5 min, infused radiotracer had moved into the third ventricle, the velum interpositum (an extension of the subarchnoid system that contains many blood vessels), the aqueduct, the mesencephalic and fourth ventricles, and the superior medullary velum (a part of the subarachnoid system that touches the mesencephalic and fourth ventricles). The CSF within both of these velae appears to empty into the quadrigeminal and ambient cisterns. Within 5 min radioactive sucrose was also found in the interpeduncular cistern. About 15% of the injected sucrose quickly left the ventricles and entered these large cisterns. In contrast to most CSF-brain interfaces, little sucrose moved from CSF into the medulla next to the lateral recesses and tissues such as the superior colliculus that lie adjacent to the large CSF cisterns. A thick, multilayered glia limitans visible on electron micrographs seemed to form a CSF-brain barrier at these interfaces. Some of the infused 14C-sucrose persisted in the perivascular spaces and walls of arteries and arterioles for more than 3.5 hr. These findings suggest that CSF may function to deliver various agents and factors to pial and parenchymal arteries and arterioles.